The long-term objectives of this application are to learn more about fundamental aspects of transcription initiation in eukaryotes. This process is strongly conserved in all eukaryotes, ranging from yeast to human. The studies will focus on protein factors discovered in the yeast, Saccharomyces cerevisiae, that control the activity of TATA-binding protein (TBP). TBP is a highly conserved protein, found throughout eukaryotes, that is essential for transcription initiation by all three nuclear RNA polymerases. The studies in this application are focused on transcription initiation by RNA polymerase II. A combination of genetic and biochemical studies have shown that two yeast transcription factors, Mot1p and Spt3p, act as negative and positive regulators, respectively, of TBP activity. Mot1p is an essential function that can remove TBP from its normal DNA binding site, a TATA box. Spt3p is a nonessential function; its activity may be to counteract Mot1p activity at particular promoters, thereby conferring promoter-specificity on TBP binding to DNA. The proposed experiments are to use genetic, molecular, and biochemical approaches to analyze Spt3p and its relationship with Mot1p, and to study how these and other related transcription factors interact with TBP to control transcription initiation. First, the interaction between Spt3p and Mot1p will be examined by several approaches, including the isolation of new mot1 mutations that affect the Mot1p-Spt3p interaction, a direct test for Mot1p-Spt3p physical interaction, and in vitro transcription experiments to attempt to define the roles of these functions and their interactions in transcription. Second, other important aspects of Spt3p will be elucidated by in vivo footprinting of an Spt3p-dependent promoter, mutational analysis of an Spt3p-dependent promoter, and the isolation and analysis of Spt3p dominant-negative mutations. Finally, other functions related to Spt3p will be studied, including three factors identified in a recent mutant hunt for new SPT genes, and factors to be identified by a screen for other Spt3p-like factors that interact with Mot1p. These studies should reveal important aspects of transcriptional control in yeast. Given the conservation between yeast and humans, the results from these studies will be applicable to transcription in humans. These studies are relevant to human disease, since altered transcription in humans has been implicated in diseases such as cancer.